1. Field of the Invention
This invention pertains to peptide inhibitors of Neutrophil activating factor induced human neutrophil chemotaxis, and more particularly to inhibitors having their sequence in part identical to that of neutrophil activating factor. These inhibitors have potential use in preventing chemotactic behavior of neutrophils in biological fluids and thereby reducing the injury that the neutrophils cause in diverse disease states such as the adult respiratory distress syndrome.
Table 1 is a list of abbreviations used for the amino acids comprising the peptides
TABLE 1 ______________________________________ A ala alanine B asx (asparagine or aspartic acid) C cys cysteine D asp aspartic acid E glu glutamic acid F phe phenylalanine G gly glycine H his histidine I ile isoleucine K lys lysine L leu leucine M met methionine N asn asparagine P pro proline Q gln glutamine R arg arginine S ser serine T thr threonine V val valine W trp tryptophan Y tyr tyrosine Z glx (glutamic acid or glutamine) ______________________________________
2. Description of Related Art
Neutrophil activating factor (NAF) is a peptide produced by stimulated human monocytes. It is chemotactic for human neutrophils, but not lymphocytes, platelets, or monocytes..sup.1-4 NAF is medically of particular interest as a potential intervention point for prevention or treatment of Adult Respiratory Distress Syndrome (ARDS).
Native NAF appears to be released in vivo in response to some forms of stress, and may be the cause of the observed neutrophil influx noted in lung fluid occurring early in the course of ARDS. Neutrophils are thought to cause lung damage which may result in death. An agent which would inhibit the chemotactic effects of NAF, without itself inducing chemotaxis, has potential application not only in ARDS but also other forms of inflammation that may be caused by NAF and in the in vitro and in vivo study of the effects of NAF.
Neutrophils have been implicated in tissue damage occurring after heart attacks. Normally employed to fight invading microorganisms, neutrophils may actually damage healthy tissue or recovering tissue because of release of substances intended to attack foreign matter. When accumulating at the site of an inflammation, neutrophils may actually block tiny blood vessels and in the heart cause severe post-heart attack tissue damage..sup.5 Several research groups are actively exploring the development of drugs to prevent neutrophils from adhering to vessel walls. Approaches include the use of antibodies to "coat" the neutrophils and drugs that prevent activation of neutrophils.
NAF is known by several different names, including NAP-1,.sup.6 MONAP,.sup.7 MCDNF,.sup.2 LYNAP 9110 and GCP..sup.4 The name interleukin-8 (IL-8) has been proposed for this molecule..sup.8
NAF has been described and partially sequenced virtually simultaneously by three laboratories..sup.2-4 Two groups.sup.2, 4 noted that the sequence was identical to that inferred from the cDNA of protein 3-10C induced in peripheral leukocytes by Staphylococcal enterotoxin A..sup.9 The full genomic sequence encoding for NAF is now known.sup.10 and the regulation of the mRNA for NAF has been investigated..sup.11,12 Tumor necrosis factor and interleukin-1 induced NAF gene transcription in monocytes after one hour of exposure, and secretion was induced between 1 and 6 hours of exposure. Interferons gamma and alpha had no effect on the gene transcription..sup.12
The function of NAF is dependent on GTP-binding proteins..sup.1 This leads to activation of protein kinase and to a transient increase in cytostolic Ca.sup.++. Surface receptors apparently are different from those of other chemotactic agents such as leukotriene B.sub.4, platelet activating factor, C5a and FMLP..sup.13
There have been conflicting reports on the ability of NAF to initiate neutrophil degranulation. Schroder et al..sup.7 reported the release of beta-glucuronidase from neutrophil primary granules while Willems et al..sup.14 showed that NAF did not induce significant release of this enzyme activity.
NAF has been isolated, sequenced and purified. Its main form has been produced from a gene synthesized, cloned and expressed in E. Coli..sup.15 Four antibodies against NAF have been produced and characterized and then used to show cross reactivity with partially homologous peptides..sup.16 All antibodies could be used in an immunoaffinity column to isolate a 10 kDa protein having NAP/IL-8 activity. This peptide, as well as two others having the same chemotactic activity, have been sequenced. The other peptides, described as variants of the main form, differ in having seven and five additional amino acids respectively at the N-terminus relative to the main form..sup.2